TGF-beta-induced interleukin-6 participates in transdifferentiation of human Tenon's fibroblasts to myofibroblasts.

Purpose To gain a better understanding of the roles of interleukins (ILs) in subconjunctival fibrosis, we investigated their expression in transforming growth factor-β1 (TGF-β1)-stimulated Tenon’s fibroblasts and examined their association with the transdifferentiation of fibroblasts to myofibroblasts. Methods After primary culture, fibroblasts derived from human Tenon’s capsule were exposed to TGF-β1. The expression of α-smooth muscle actin (α-SMA) protein was assessed by western immunoblots and immunofluorescence. The mRNA levels of various ILs were also evaluated by multiplex reverse transcription (RT)-PCR. Using the small interfering RNAs (siRNAs) specific for IL-6 and IL-11 and the promoter deletion assay, the contributions of IL-6 and IL-11 to TGF-β1-induced induction of α-SMA were determined. Results In human Tenon’s fibroblasts, TGF-β1 stimulated the expression of α-SMA protein determined by western blot analysis and also increased the mRNA levels of IL-6 and IL-11 determined by multiplex RT-PCR. On the western immunoblots and immunofluorescence, the increased expression of α-SMA was attenuated only by the siRNAs specific for IL-6 but not by the siRNAs specific for IL-11. When the activator protein-1 binding sites of the IL-6 promoter region were deleted, the stimulation effects of TGF-β1 decreased. Conclusions Our data show that autocrine IL-6 may participate in the TGF-β1-induced transdifferentiation of human Tenon’s fibroblasts to myofibroblasts, which is known to be an essential step for subconjunctival fibrosis.

In the present study, we were interested in investigating the relationship between inflammation and fibrosis in human Tenon's fibroblasts. In lung and heart, certain types of inflammation recruit and stimulate fibroblasts in a TGF-βdependent manner [15][16][17][18]. These activated fibroblasts then transdifferentiate to myofibroblasts that produce extracellular matrix (ECM); these contractile cells ultimately cause extensive fibrosis. In this study we investigated which of the proinflammatory cytokines of the interleukin (IL) family are System, Minneapolis, MN) after 24 h of serum starvation in serum-free DMEM.
RNA interference assay: siRNA molecules targeting IL-6 and IL-11 mRNA were purchased from Ambion, Inc. (Austin, TX). The RNA duplex against IL-6 had the sequence 5'-GGA CAU GAC AAC UCA UCU CTT-3' (sense) and 5'-GAG AUG AGU UGU CAU GUC CTG-3' (antisense); and the RNA duplex against IL-11 had the sequence 5'-GCA ACA UGG UGC AUC UGU GTT-3' (sense) and 5'-CAC AGA UGC ACC AUG UUG CTT-3' (antisense). siRNAs were delivered into cells according to the manufacturer's instructions. Briefly, the diluted transfection reagent (siPORT Amine; Ambion) was mixed with the diluted siRNA to allow the formation of transfection complexes. This mixture with a final RNA concentration of 20 nM was then dispensed onto the cells and incubated for 24 h.
Cells were transfected with an equal amount of reporter plasmid using Lipofectamine 2000 (Invitrogen). The luciferase activities were determined using a luciferase assay system (Promega, Co., Madison, WI), following the manufacturer's instruction. Briefly, the 96-well plate containing 20 μl of cell lysate per well was placed into the luminometer with injector. The injector added 100 μl of luciferase assay reagent per well, then the well was read immediately. The plate was advanced to the next well for a repeat of the inject-then-read process. The typical delay time was 2 s and the typical read time was 10 s. Data analysis: Densitometric data are expressed as means ± standard deviation (SD). Data were compared with Kruskal-Wallis one-way analysis of variance and Mann-Whitney U test using the SPSS program for Windows, version 12.0.1 (SPSS Inc., Chicago, IL). A p value less than 0.05 was considered statistically significant.

RESULTS
Transforming growth factor-β1 induces transdifferentiation of fibroblasts to myofibroblasts: To evaluate the effect of TGF-β1 on the expression of α-SMA, we measured α-SMA protein levels in human Tenon's fibroblasts stimulated with different concentrations of TGF-β1 for different durations. Exposure of fibroblasts to TGF-β1 resulted in a dose-and time-dependent increase in expression of α-SMA ( Figure 1). TGF-β1 treatment (10 ng/ml) for 72 h resulted in a significant increase (p<0.001) in α-SMA expression. Because the presence of α-SMA is a phenotypic hallmark of myofobroblasts, this finding indicates that TGF-β1 induced the transdifferentiation of fibroblasts to myofibroblasts.
Transforming growth factor-β1-induced expression of αsmooth muscle actin is reduced by IL-6-specific small interfering RNAs: To more directly address the possible involvement of IL-6 and IL-11 in TGF-β1-induced myofibroblast transdifferentiation, we evaluated the effects of IL-6 and IL-11 knockdown using specific siRNAs. Since the accumulation of ECM proteins is an indicator of Figure 1. Representative western blot bands for α-smooth muscle actin. A: Dose-response effects: fibroblasts were exposed to various concentrations of transforming growth factor (TGF)-β1 for 72 h. B: Time-response effects: fibroblasts were incubated with or without 10 ng/ml of TGF-β1 for up to 6 days. β-actin was used as an internal control. Densitimetric data represent the mean±SD of results from three independent experiments. myofibroblast transdifferentiation, we studied the effect of IL-6-and IL-11-specific siRNAs on TGF-β1-induced expression of the ECM component α-SMA. Treatment with IL-6-specific siRNAs strongly inhibited the TGF-β1-induced α-SMA expression ( Figure 3A), whereas IL-11-specific siRNAs had little effect ( Figure 3B).
To confirm this finding, we assessed the effects of IL-6and IL-11-specific siRNAs on TGF-β1-induced α-SMA upregulation by immunofluorescent staining and obtained similar results with western immunoblot analysis (Figure 4). Compared to untreated control fibroblasts, TGF-β1stimulated fibroblasts showed a strong cytoplasmic α-SMA signal with numerous intracellular stress fibers ( Figure 4B). Fibroblasts transfected with IL-6-specific siRNAs showed a significant reduction in the effect of TGF-β1 on α-SMA expression ( Figure 4D); but cytoplasmic staining for α-SMA was little diminished in the presence of IL-11-specific siRNAs ( Figure 4F). These data demonstrate that knockdown of IL-6 reduces the ability of TGF-β1 to induce α-SMA expression.
Activator protein-1 activity at the IL-6 promoter in the presence of transforming growth factor-β1: To determine whether TGF-β1-mediated myofibroblast transdifferentiation might be related to AP-1-binding site-dependent effects on IL-6 transcription, we used wild-type and mutant IL-6 promoter-reporter constructs to analyze IL-6 promoter activity ( Figure 5). TGF-β1 increased the promoter activity of the wild-type-reported plasmid about 2.5-fold above the notreatment control (p<0.001). However when the 3' or 5' end of the AP-1-binding site of the IL-6 promoter was deleted, the increased promoter activity by TGF-β1 was significantly attenuated (p<0.001). Our results indicate that the effect of TGF-β1 on IL-6 expression is dependent on the AP-1-binding site of the IL-6 promoter region and suggest that TGF-β1- induced myofibroblast transdifferentiation is dependent, at least in part, on TGF-β1-induced IL-6 expression. Figure 3. RNA interference assay for IL-6 and IL-11. After administering small interfering RNAs targeting IL-6 (A) and IL-11 (B), fibroblasts were exposed to 10 ng/ml transforming growth factor (TGF)-β1 for 72 h. Then, the expression of α-smooth muscle actin (α-SMA) was evaluated using western blots. Densitimetric data represent the mean±SD of results from three independent experiments. The asterisks refer to a significant difference compared to TGF-β treatment only (p<0.001).

DISCUSSION
Transdifferentiation of fibroblasts to myofibroblasts is at the core of the fibrotic process. Although the fibrosis caused by these activated fibroblasts is essential for natural wound healing, it can also result in excessive scarring, which is one of the most common causes of surgical failure after various ocular surgeries, including glaucoma-filtering procedures [1][2][3][4][5][6]. Although adjunctive antimetabolites can suppress the proliferation of fibroblasts and reduce subconjunctival fibrosis, the use of these compounds may result in serious complications; thus a new treatment strategy to prevent excessive fibrosis is required.
In various organs, including the lung and heart, inflammation is known to result in the development of fibrosis; TGF-β seems to be at the center of this process [15][16][17][18]. At the ocular surface, TGF-β plays a key role as a potent fibrogenic cytokine [25][26][27][28][29]. It is thought to stimulate the chemotaxis and transdifferentiation of fibroblasts, resulting in the overproduction of collagen, fibronectin, and other ECM components. Furthermore, TGF-β reduces the degradation of synthesized ECM through suppression of the activity of matrix metalloproteases and the activation of protease inhibitors. IL-6 is essentially a chemoattractant and stimulator of lymphocytes [30][31][32][33]. It also functions as a pleiotropic mediator in the acute-phase response and stimulates the differentiation and proliferation of various target cells [33]. IL-6 exerts its effects through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway [34]. Because the IL-6 receptor does not have intrinsic tyrosine kinase activity, when IL-6 binds to the extracellular ligand-binding domain of its receptor, members of the JAK family are activated and phosphorylate the tyrosine residues of the IL-6 receptor, which then act as docking sites for the SH2 domains of STATs. IL-6-activated T lymphocytes then secrete TGF-β and trigger the fibrotic cascade.
Consistent with a previous report that IL-6 participates directly in the transdifferentiation of Tenon's capsule fibroblasts to myofibroblasts [35], in the present study we verified the presence of autocrine IL-6 from activated Tenon's capsule fibroblasts. Figure 5. Promoter deletion assay for interleukin-6 (IL-6). Three recombinant plasmids, in which different fragments of the activator protein-1 (AP-1)-binding site on the human IL-6 promoter were deleted, were used. After transfection with each plasmid, fibroblasts were incubated with 10 ng/ml transforming growth factor (TGF)-β1 for 72 h. The luciferase activity was then determined and expressed as the percent of the no-TGF-β1 treatment control. Data represent the mean±SD of results from three independent experiments. The asterisks refer to a significant difference (p<0.001) compared to the wild type.
We found that the transcription of IL-6 and IL-11 was increased by TGF-β1 stimulation in human Tenon's fibroblasts, and that this was accompanied by the upregulated expression of α-SMA. When IL-6-specific siRNAs were used, the TGF-β1-stimulated increase in expression of α-SMA was blocked, but this did not occur when IL-11-specific siRNAs were used. Our results indicate that autocrine IL-6 produced as a result of TGF-β-stimulation of human Tenon's fibroblasts stimulates the transdifferentiation of these fibroblasts to myofibroblasts, which is thought to be essential for subconjunctival fibrosis. Modulation of autocrine IL-6 production might be useful as a novel therapeutic strategy for controlling postoperative subconjunctival fibrosis.